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Bioresource Technology

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Shifting product spectrum by pH adjustment during long-term continuousanaerobic fermentation of food waste

Kai Fenga, Huan Lia,b,⁎, Chengzhi Zhengc

a Shenzhen Engineering Research Laboratory for Sludge and Food Waste Treatment and Resource Recovery, Graduate School at Shenzhen, Tsinghua University, Shenzhen518055, ChinabGuangdong Engineering Research Center of Urban Water Cycle and Environment Safety, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Chinac Technical Department of Rocktek, Rocktek Limited Liability Company, Wuhan 430223, China

A R T I C L E I N F O

Keywords:Anaerobic fermentationFood wasteMicrobial communityMetabolic pathway

A B S T R A C T

Anaerobic fermentation is widely used to recover different products from food waste, and in this study, theevolution of fermentation products and microbial community along with pH variation was investigated thor-oughly using four long-term reactors. Lactic fermentation dominated the system at pH 3.2–4.5 with lactic acidconcentration of 5.7–13.5 g/L, and Lactobacillus was the superior sort. Bifidobacteria increased significantly at pH4.5, resulting in the increase of acetic acid. Butyric acid fermentation was observed at pH 4.7–5.0.Bifidobacterium, Lactobacillus, and Olsenella were still dominant, but the lactic acid produced by them wasconverted to volatile fatty acids (VFAs) rapidly by Megasphaera, Caproiciproducens, Solobacteria, etc. Mixed acidfermentation occurred at pH 6.0 with the highest concentration 14.2 g/L of VFAs, and the dominant Prevotellaand Megasphaera converted substrates to VFAs directly. On the whole, pH 4.5 and 4.7 led to the highest hy-drolysis rate of 50% and acidification rate of 45%.

1. Introduction

Food waste (FW), which is a part of municipal solid waste, hasenormous potential for energy and resource recovery because it is richin nutrients and organics. Recently, recovering value-added chemicalsfrom FW through anaerobic fermentation has attracted a wide attentiondue to its advantages of waste minimization and simultaneous utiliza-tion. During anaerobic fermentation, bio-based materials can be har-vested, including hydrogen, methane, volatile fatty acids (VFAs), lacticacid, ethanol, etc. (Jang et al., 2012) Hydrogen and methane are the gasproducts, which can be used as the substitutes of fossil fuels. VFAs,Lactic acid and ethanol are the common liquid-phase products, whichcan be used as the basic materials in many fields including textile,medical, leather, plastics and energy industries (Castillo Martinez et al.,2013). In addition, lactic, VFAs or their mixture can be used as thecarbon source of biological denitrification in wastewater treatment(Zhang et al., 2017).

There are two basic technical pathways for anaerobic fermentationof FW, i.e. pure culture and mixed culture. The former inoculates FWusing only a sort of microorganism, aiming to homogeneous fermen-tation with high yields of target products. For example, Lactobacilluswas used for lactic acid production, and Yeast was adopted to produce

ethanol. Compared with this pure culture, mixed culture with highmicrobial diversity is more suitable for bioprocesses under non-sterileconditions. This mode is resilient to environmental fluctuation due tomicrobial functional redundancy, and it allows continuous operationwithout strain degeneration (Temudo et al., 2007). In addition, mixedculture fermentation is less costly than pure culture fermentation inindustrial scale (Esquivel-Elizondo et al., 2017). These advantagesmake it become the main method for decomposition or conversion ofcomplex organic wastes like FW.

In a fermentation system with mixed culture, pH is a key factordetermining fermentation type and yields of target products becausemicrobial community structure, metabolic pathway, and enzyme ac-tivity are all affected by pH (Tang et al., 2017). However, there is noconsistent conclusion on the composition of fermentation products ofFW under different pH conditions (Zhou et al., 2018). Wu et al. (2015b)found that the main fermentation product of fruit and vegetable wasteat pH 4.0 was lactic acid with an average content of 60.4% (based onmass), while Wu et al. (2017) found in another experiment that ethanolcontent was 88.8% at pH 4.0. Tang et al. (2017) found that lactic acidhad the highest yield at pH 5.0, whereas Wang et al. (2014) found thatbutyric acid was the main product at pH 5.0, with a relative content ofup to 80%. Jiang et al. (2013) found that butyric acid was the majority

https://doi.org/10.1016/j.biortech.2018.09.035Received 5 August 2018; Received in revised form 4 September 2018; Accepted 6 September 2018

⁎ Corresponding author at: Room 2113, Building of Energy and Environment, Tsinghua Campus, University Town, Shenzhen, China.E-mail address: li.huan@sz.tsinghua.edu.cn (H. Li).

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Available online 08 September 20180960-8524/ © 2018 Elsevier Ltd. All rights reserved.

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at pH 6.0, but Bengtsson et al. (2008) reported that the ratios of aceticacid, propionic acid and butyric acid changed from 51:19:24 to31:41:10 (based on mass) when pH rose from 5.3 to 6.0. These incon-sistent conclusions could be attributed to different substrate composi-tions, redox potentials, and organic load rates (OLRs). However, theexisting studies rarely took all the VFAs, lactic acid, ethanol and hy-drogen into account when discussing fermentation types of FW. More-over, many studies were carried out in batch mode in a short period,which possibly screened the real variation during long-term anaerobicfermentation of FW. Furthermore, the fundamental mechanism forthese different results is the various metabolic pathways of functionalmicroorganisms degrading organic substrates, but the evolution ofmetabolic pathways had not been considered thoroughly during anae-robic fermentation of FW under different pH conditions.

To reveal the intrinsic effect of different pH on fermentation pro-ducts (hydrogen, ethanol, lactate, VFAs), four continuous fermentationreactors were operated in a long term using FW as the feedstock andanaerobic sludge as the inoculum. The pH condition was controlled atdifferent levels, and the corresponding fermentation products and mi-crobial community structure were investigated thoroughly.Particularly, metabolic pathways related to different pH conditionswere fully discussed. At the end, hydrolysis rates and acidification rateswere analyzed, aiming to obtain the optimal conditions for differentapplication scenarios concerning FW utilization. The results were ex-pected to provide an overall view on anaerobic fermentation of FWbased on mixed culture.

2. Materials and methods

2.1. Inoculum and substrate

The inoculum was the anaerobic sludge collected from an anaerobicdigester in the laboratory, which was readily operated for more thanone year (Li et al., 2018). Simulated FW was used as the feedstock(substrate) to keep repeatable conditions, and the FW was consisted of36% vegetables, 34% rice, 8% meat, 6% noodle, 5% egg, 4% tofu, 4%edible oil, and 3% condiments. The vegetables contained 37% cabbage,33% potato, 12% carrot, 16% onion and 2% garlic. The condimentscontained 13% salt, 30% soy sauce, 4% pepper, 8% monosodium glu-tamate, 30% thick broad-bean sauce, 15% oyster sauce. These compo-nents were mixed and cooked for 30min in an auto rice cooker, andthen crushed using a FW disposer. The prepared samples were stored ina refrigerator at 4 °C. The characteristics of inoculum and substrate areshown in Table 1.

2.2. Fermentation reactors

Four continuous stirred tank reactor (RTK-CSTR, Rocktek, China)were used to investigate the influence of pH on anaerobic fermentationof FW (Fig. 1). Each one had a working volume of 3 L, which wasequipped with an auto stirrer, a pH controller, and a temperaturecontroller. The stirrer with flexible height was connected to a variable

speed motor, and the stirring speed was fixed at 40 rpm. The pH wasadjusted using a pH meter and a peristaltic pump that can add 2.0 mol/L NaOH solution or 1.0 mol/L HCl to the reactor. The temperature waskept at 35 °C using an external circulating water bath. A gas flow meter(RTK-SGMC, Rocktek, China) was employed to record biogas yieldautomatically, and a gas bag was installed on the outlet of the flowmeter to collect biogas for compositional analysis. The feed pipe wasinserted into fermentation liquid to prevent gas leakage and oxygenentry during feeding process.

The four reactors were all fed and drawn off once a day, and thedischarged effluent was used for analysis. For all the four reactors, thesolid retention time (SRT) was set at 4 d, the TS of feedstock was ad-justed to 5%, and correspondingly, the OLR was 11.8 g VS/(L d). ThepH of one reactor was uncontrolled originally (R1), and the pH valuesof the other three reactors were controlled at 4.0 (R2), 4.2, 4.5, 4.7 (R3with three stages), 5.0 and 6.0 (R4 with two stages), respectively.

2.3. Fermentation degree

Fermentation degrees can be expressed using hydrolysis rate (HR)and acidogenic rate (AR). HR represents the dissolution degree of or-ganic matter in solid phase, and AR indicates the total yield of targetproducts including lactic acid, ethanol and VFAs. They were calculatedbased on COD or TOC, as shown in Eqs. (1)–(4).

= ×HR COD /TCOD 100%COD S 0 (1)

= ×HR TOC /TOC 100%TOC S 0 (2)

= ×AR COD /TCOD 100%COD A 0 (3)

= ×AR TOC /TOC 100%TOC A 0 (4)

where, CODS was the soluble COD of fermentation effluent, TCOD0 wasthe total COD of feedstock, TOCS was the soluble TOC of fermentationeffluent, TOC0 was the initial TOC of feedstock, CODA was the calcu-lated COD of fermentation products including VFAs, ethanol, and lacticacid, and TOCA was the TOC of fermentation products including VFAs,ethanol, and lactic acid.

2.4. Analytical procedures

Fermentation effluent was sampled and sent to Majorbio Inc.,

Table 1Characteristics of anaerobic sludge and food waste.

items Inoculum sludge Food waste

TS (%) 18.5 ± 0.3 14.0–20.0VS (%) 9.9 ± 0.2 13.3–19.0VS/TS (%) 53.3 ± 0.6 95.0 ± 0.03SCOD (mg/L) 5737 ± 94 7042 ± 406*

TCOD (mg/L) – 58333 ± 1172*

NH3-N (mg/L) 1798 ± 34 20 ± 7*

C/N 7.3 21.3pH 7.2 ± 0.1 5.2 ± 0.1

* After diluted to 5% (TS).

Fig. 1. Schematic diagram of the fermentation reactor.

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Shanghai, China for high-throughput sequencing. DNA was extractedusing PowerSoil® DNA kit (Mo Bio Laboratories Inc., Carlsbad, U.S.A.)and then amplified using PCR amplifier (GeneAmp®9700, ABI, U.S.A.).The primer pairs were 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and806R (5′-GGACTACHVGGGTWTCTAAT-3′). The amplification productswere purified and then sequenced (MiSeq, Illumina, U.S.A.). The resultswere analyzed using the software platform QIIME 1.17.

Elementary compositions of the FW and the inoculum were de-termined by an elemental analyzer (vario EL cube, Elementar, Hanau,Germany). To analyze soluble parameters, samples were first cen-trifuged at 5800g for 60min, the supernatant was filtered by mem-branes with mesh size of 0.45 μm, and the filtrate was used for analyses.TS, VS, total alkalinity (TA), TAN, and SCOD were determined ac-cording to standard methods (MEP, 2002). The pH was measured by anonline monitor (CN113C, CNIC, China) with a pH detector (CN11D,CNIC, China), and the ORP was measured by a digital meter (PHS-3C,INESA, China) with an ORP detector (501, INESA, China). Ethanol andVFAs (acetic acid, propionic acid, butyric acid, valeric acid) weremeasured using a gas chromatograph (GC-2014, Shimadzu, Japan)equipped with a capillary column (Inertcap wax30m×0.25mm×0.25 μm) and a flame ionization detector. The lacticacid was determined by a high-performance liquid chromatography(Prominence UFLC, Shimadzu, Japan) equipped with an InertSustain®C18 column (5 μm, 25 cm×4.6mm) and an ultraviolet (206 nm) de-tector, while 85% 20mmol/L KH2PO4 and 15% methanol were used aseluent at a rate of 0.7ml/min. The biogas flow was measured by a wetgas flow meter (RTK-SGMC, Rocktek, China). The composition of biogaswas analyzed using a gas chromatograph (MGC-7850S, Jing He, China)equipped with a thermal conductivity detector.

2.5. Statistical analysis

According to the operational setting, seven pH conditions weretested in the four fermentation reactors, including uncontrolled pH inR1 (which finally kept stable at 3.2), pH 4.0 in R2, pH 4.2, 4.5 and 4.7in R3, pH 5.0 and 6.0 in R4. A special phenomenon was found at thebeginning stage of R3, i.e., the pH increased from 4.5 to 4.7 sponta-neously because the initial reactor was equipped with a pH controller ofonly adding alkali. The data collected from the stable stages, where theconcentrations of main fermentation products had relative standarddeviations less than 20%, were used for analyses and comparison, asshown in Table 2. These data were expressed as mean and standarddeviation, and significance analyses were adopted in the comparisonusing Microsoft Excel.

3. Results and discussion

The four reactors were operated under different pH conditions for150 d (R1 and R2) or 190 d (R3 and R4), and the gaseous and liquidproducts were measured including H2, CO2, CH4, ethanol, lactic acidand VFAs. However, no CH4 was observed during these processes

except for the initial several days. All the daily data were recorded inFig. 2, and the average values in stable stages corresponding to differentpH conditions were given in Table 3, together with their standard de-viations. Fermentation products was sourced from metabolism offunctional microorganisms, and the composition of fermentation pro-ducts was determined by microbial community structure in fermenta-tion systems. For example, Lactobacillus can produce lactic acid andethanol (Castillo Martinez et al., 2013), Bifidobacterium can producelactic acid and acetic acid (Pokusaeva et al., 2011), and Megasphaeracan produce acetic acid, propionic acid, butyric acid, valeric acid andhydrogen (Weimer & Moen, 2013). In addition, the same species couldoutput different metabolites due to the change of metabolic pathwaysunder different environmental conditions. Hence, microbial analysiswas carried out at different pH values (Fig. 3). Compared with the in-oculum, the fermentation reactors had different microbial communitystructures, indicating the evolution of fermentation types.

3.1. Lactic acid fermentation at pH 3.2–4.5

In the first 10 d of R1 without pH control [Fig. 2(a)], pH decreasedgradually and then maintained at 3.2. Lactic acid was the main product,which accounted for 86.5% of the total products by mass. The otherproducts like ethanol and VFAs were very rare. Jiang et al. (2013) re-ported that the pH decreased to 3.6 without any intervention, thecorresponding yields of ethanol and VFAs were very low, but they didnot measure lactic acid. The gas products (H2 and CO2) also decreasedalong with the decline of pH, and no more gas generated after the 9thday. This implied that homolactic fermentation (Eq. (5)) dominated thesystem through Embden-Meyerhof-Parnas (EMP) pathway (CastilloMartinez et al., 2013). In fact, Lactobacillus was the dominant bacterium(97.55%, indicating the relative abundance and the same below), whichhas high endurance capability to low pH (Fuess et al., 2018; Itoh et al.,2012). However, most of other fermentative bacteria cannot survivefrom low pH (< 4.0) and un-dissociated VFAs that can enter into mi-crobial cells and inhibit their metabolism (Elbeshbishy et al., 2017).Thus, the production of VFAs, ethanol and H2 was inhibited. Moreover,some lactic acid bacteria (LAB) can inhibit the activity of hydrogen-producing bacteria (HPB) by producing reactive oxygen species (e.g.,hydrogen peroxide) and secreting polypeptide antibiotics such as bac-teriocins (Elbeshbishy et al., 2017).

In the R2 of pH 4.0 [Fig. 2(b)], lactic acid was still the main product,but its concentration was 2–3 times higher than that in the R1 (pH 3.2).Simultaneously, the concentration of ethanol increased to1.57 ± 0.20 g/L, the yield of CO2 reached 16.9 ± 2.9ml/g VS, butthat of H2 remained at 1.7 ± 0.6 ml/g VS. Hence, in addition tohomolactic fermentation, heterolactic fermentation with ethanol oracetic acid as the co-products occurred in the system (Castillo Martinezet al., 2013; Pokusaeva et al., 2011). Correspondingly, Lactobacillus wasstill dominant (93.87%), but the other genera increased, includingAcetobacter (0.98%), Bifidobacterium (0.93%), Megasphaera (0.80%),and Pseudomonas (0.52%). Lactobacillus can produce lactic acid andethanol (Eq. (6)) through Phosphoketolase (PK) pathway (CastilloMartinez et al., 2013; Sonderegger et al., 2004), and Bifidobacterium canproduce lactic and acetic acid (Eq. (7)) through Bifidus pathway (Donget al., 2000; Kandler, 1983; Pokusaeva et al., 2011). Some species ofAcetobacter can produce acetic acid using glucose (Chen & Wang, 2017),Megasphaera can produce H2 and VFAs using lactic acid and glucose(Moreno-Andrade et al., 2015; Weimer & Moen, 2013), and Pseudo-monas can also produce acetic acid, butyric acid and H2 (Guo et al.,2008). As a result, the production of ethanol and VFAs increased sig-nificantly. Similarly, Wu et al. (2015b) obtained lactic acid with aconcentration of 17.22 g/L during anaerobic fermentation of fruit andvegetable wastes, and Tang et al. (2017) found that lactic acid with aconcentration of 19.0 g/L was the majority of fermentation products.

The second stage of R3 was controlled at pH 4.2 [Fig. 2(c)]. The twoconditions of pH 4.2 and 4.0 had similar composition of fermentation

Table 2Stage division and sampling data for microbial analysis corresponding to dif-ferent pH conditions.

pH Reactor RunningPeriod (d)

Stable stage forproducts analyses(d)

Sampling date formicrobial analysis

3.2 R1 1–150 85–150 91, 103, 114,4.0 R2 1–150 81–150 103, 116, 1484.2 R3 67–119 89–119 1004.5–4.7 R3 1–66 / /4.5 R3 151–191 155–191 177, 185, 1914.7 R3 120–150 139–150 1485.0 R4 1–150 27– 50, 77–91 29, 90, 1186.0 R4 151–191 175–191 187, 189, 191

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products and similar structure of microbial community. The relativeabundance of Bifidobacterium, Acetobacter and Pseudomonas increasedfurther, while that of Megasphaera reduced a little. Accordingly, theyield of H2 decreased slightly. When the pH of R3 was controlled at 4.5accurately at the third stage [Fig. 2(c)], the concentration of lactic acidwas similar with that under 4.2, but the concentration of acetic andbutyric acid increased greatly. Moreover, no much H2 was producedalong with the production of acetic and butyric acid. This implied thatthe system still tended to homolactic fermentation, but other fermen-tation types amplified further. The acetic acid could source from het-erolactic fermentation through Bifidus pathway, as shown in Eq. (7)(Castillo Martinez et al., 2013; Pokusaeva et al., 2011). Correspond-ingly, the relative abundance of Lactobacillus decreased to 65.54%,while that of Bifidobacterium increased greatly to 25.38%. Moreover,

Megasphaera increased to 2.16% with much more production of VFAsand H2. Compared with the condition of pH 4.2, the condition of pH 4.5had higher concentrations of acetic and butyric acid and similar yield ofH2. It was deduced that some H2 was consumed on the production ofvaleric and propionic acid.

The results proved that lactic fermentation was stable in the pHrange of 3.2–4.5. Zheng et al. (2015) also reported good stability oflactic fermentation at pH 4.0, and a slight fluctuation of pH had almostno influence on fermentation products. On the whole, there should bethree key reactions of lactic acid fermentation at pH 3.2–4.5, as shownin Eqs. (5)–(7). Moreover, when pH increased from 3.2 to 4.5, thefermentation types changed from homolactic fermentation throughEMP to heterolactic fermentation through PK and Bifidus pathways.Besides the main reactions, there were still some side reactions, which

Fig. 2. Fermentation products under different pH conditions (a: 3.2; b: 4.0; c: 4.2 and 4.5; d: 5.0 and 6.0).

Table 3Fermentation products under different pH conditions (mean ± standard error).

pH units 3.2 4.0 4.2 4.5 4.7 5.0 6.0

Lactic acid mg/L 5681 ± 769 13366 ± 1071 13549 ± 1414 12466 ± 1722 449 ± 282 623 ± 422 353 ± 401mg/g VS 481 ± 65 1133 ± 91 1148 ± 120 1056 ± 146 38 ± 24 53 ± 36 30 ± 34

Ethanol mg/L 517 ± 132 1565 ± 204 1487 ± 161 735 ± 108 1516 ± 267 1291 ± 361 499 ± 93mg/g VS 44 ± 11 133 ± 17 126 ± 14 62 ± 9 128 ± 23 109 ± 31 42 ± 8

Acetic acid mg/L 328 ± 82 567 ± 86 562 ± 69 5487 ± 781 3025 ± 290 4072 ± 482 2448 ± 284mg/g VS 28 ± 7 48 ± 7 48 ± 6 465 ± 66 256 ± 25 345 ± 41 207 ± 24

Propionic acid mg/L 24 ± 17 285 ± 88 283 ± 67 844 ± 485 713 ± 683 91 ± 89 1343 ± 126mg/g VS 2 ± 1 24 ± 7 24 ± 6 72 ± 41 60 ± 58 8 ± 8 114 ± 11

Butyric acid mg/L 14 ± 29 606 ± 137 554 ± 116 2035 ± 418 8784 ± 992 4066 ± 1220 5992 ± 658mg/g VS 1 ± 2 51 ± 12 47 ± 10 172 ± 35 744 ± 84 345 ± 103 508 ± 56

Valeric acid mg/L 5 ± 14 219 ± 15 283 ± 82 414 ± 160 1201 ± 344 16 ± 34 4376 ± 704mg/g VS 0.4 ± 1 19 ± 1 24 ± 7 35 ± 14 102 ± 29 1 ± 3 371 ± 60

Total acids & ethanol mg/L 6570 ± 846 16608 ± 1129 16718 ± 1806 21981 ± 1175 15687 ± 345 10072 ± 831 15011 ± 1944mg/g VS 557 ± 72 1407 ± 96 1417 ± 153 1863 ± 100 1329 ± 29 861 ± 70 1272 ± 165

H2 ml/gVS / 1.7 ± 0.5 0.4 ± 0.1 1.9 ± 0.5 27.8 ± 1.9 62.4 ± 12.6 29.5 ± 3.0CO2 ml/gVS / 16.9 ± 2.9 13.3 ± 2.6 17.7 ± 5.1 57.1 ± 3.2 82.5 ± 11.6 64.8 ± 6.9SCOD mg/L 17554 ± 1860 24772 ± 1634 25175 ± 2335 29346 ± 1731 29377 ± 808 27105 ± 1966 27861 ± 1293TOC mg/L 6639 ± 477 8957 ± 443 9000 ± 401 11254 ± 523 9258 ± 76 8471 ± 311 9897 ± 110TAN mg/L 89 ± 8 86 ± 10 82 ± 15 35 ± 8 38 ± 9 33 ± 4 20 ± 6ORP mV −277 ± 40 −401 ± 10 −388 ± 7 −451 ± 8 −479 ± 7 −510 ± 5 −504 ± 5

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contributed to the production of ethanol, VFAs and H2. Accordingly,lactic acid was always the major product, but the other by-products likeethanol and VFAs increased gradually along with pH increase.

→C H O 2CH CHOHCOOH6 12 6 3 (5)

→ + +C H O CH CHOHCOOH CO CH CH OH6 12 6 3 2 3 2 (6)

→ +C H O CH CHOHCOOH 1.5 CH COOH6 12 6 3 3 (7)

3.2. Butyric acid fermentation at pH 4.7–5.0

At the first and the third stage of R3, the pH increased sponta-neously from 4.5 to 4.7. This phenomenon had not been reported be-fore. The first stage was operated with only artificial NaOH addition.Along with the pH rise at the 30th and 47th day, butyric acid and H2

increased, while lactic and acetic acid decreased. This suggested thatbutyric acid and H2 could be sourced from the degradation of lactic acid(Baghchehsaraee et al., 2009; Kim et al., 2012) and the consumption ofH+ (Fuess et al., 2018) as lactic acid has a lower dissociation constant(pKa 3.86 at 25 °C) than butyric acid (pKa 4.82 at 25 °C). Indeed, at thethird stage of R3 (pH 4.7), butyric acid was the most (8.78 ± 0.99 g/L), while lactic acid decreased to a very low level. Compared with thestage at pH 4.5, the relative abundance of Lactobacillus decreased to15.39% at the stage of pH 4.7, while those of Bifidobacterium andMegasphaera increased further to 42.36% and 24.26%, respectively. Thelatter two sorts were ever found to be active at pH around 4.7 (Donget al., 2000; Weimer & Moen, 2013). In addition, Olsenella (15.86%)became a key sort, which is aerotolerant and can anaerobically producelactic acid (Olsen et al., 1991). Lactobacillus, Bifidobacterium and Olse-nella can produce lactic acid, ethanol and acetic acid using glucose, andMegasphaera can not only produce butyric and H2 from glucose, but alsoutilize lactic acid and acetic acid to produce butyric acid and H2 (Eqs.(8) and (9) (Fuess et al., 2018; Hino & Kuroda, 1993; Saady, 2013;Weimer & Moen, 2013). Consequently, the concentration of acetic aciddecreased, while that of butyric acid increased to the maximum.

The pH was controlled at 5.0 at the first and third stage of R4[Fig. 2(d)]. Under this condition, butyric and acetic acid were the mainliquid products, and H2 production reached the maximum67.0 ± 8.3ml/g VS with a content of 43.7% in biogas. H2 production isusually related to butyric and acetic acid production from glucose de-gradation (Fuess et al., 2018; Yun et al., 2018). In addition to thispathway, butyric acid and H2 produced at this stage should partly comefrom the degradation of lactic acid. Indeed, lactic acid increased to8.53 g/L at most on the 13th to the 19th day, and then decreased on the

20th to the 25th day. An opposite trend occurred at the production ofbutyric acid and H2, accompanied with a pH rise to 5.1. The highconcentration of lactic acid on the 92nd to the 111st day and the 122ndto 135th day was contributed to an occasional increase in OLR due tooperational fault. Temudo et al. (2007) verified that a sudden rise ofsubstrate concentration or pH variation increased the production oflactic acid. On the whole, the concentrations of butyric and acetic acidfluctuated in a limited range, and the same phenomenon at pH 5.0 wasalso reported by Zheng et al. (2015). In the microbial community, LABwas still dominant including Lactobacillus, Bifidobacterium, and Olse-nella, but some acid producing bacteria increased significantly likeCaproiciproducens (9.88%), Solobacterium (5.84%), Eubacteria nodatum(3.77%). These sorts can convert glucose to VFAs and hydrogen (Hillet al., 1987; Kageyama & Benno, 2000; Kim et al., 2015). The relativeabundance of Olsenella reached 21.43%, which could also help hy-drogen production (Li et al., 2011). In addition, these microorganismsthat directly use glucose to produce butyric acid, acetic acid and hy-drogen should metabolize through EMP pathway because only thispathway convert pyruvate to acetyl-CoA with hydrogen production.

In the above ways, lactic acid was still produced by LAB, but it wasimmediately utilized to produce butyric acid. Simultaneously, glucosewas also directly degraded to produce different VFAs. Thus, the mainreactions can be concluded as Eqs. (5)–(9) plus Eqs. (10)–(12) (Zhouet al., 2018).

+ → +

+ +

4CH CHOHCOOH 2CH COOH 3CH CH CH COOH 4CO

2H 2H O3 3 3 2 2 2

2 2 (8)

→ + +2CH CHOHCOOH CH CH CH COOH 2CO 2H3 3 2 2 2 2 (9)

→ + +C H O CH CH CH COOH 2CO 2H6 12 6 3 2 2 2 2 (10)

+ → + +C H O 2H O 2CH COOH 2CO 4H6 12 6 2 3 2 2 (11)

+ → +CH CH CH COOH 2H O 2CH COOH 2H3 2 2 2 3 2 (12)

3.3. Mixed acid fermentation under pH 6.0

At the fourth stage of R4 [Fig. 2(d)], the pH was increased to 6.0.After the system became stable, the concentrations of lactic acid andethanol were quite low, while butyric acid, valeric acid, acetic acid, andpropionic acid became the main products, indicating a feature of mixedacid fermentation. Similar result was also reported by Yu et al. (2018).Particularly, the concentrations of propionic acid and valeric acid in-creased to 1.34 ± 0.13 and 4.38 ± 0.70 g/L, respectively, but theyield of H2 decreased compared with that at pH 5.0. The generation ofvaleric acid and propionic acid could consume some H2 (Eq. (15))(Saady, 2013). In the microbial community, Lactobacillus almost dis-appeared, and the relative abundances of Bifidobacterium and Olsenellaalso decreased to about 5%. Prevotella (57.47%) and Megasphaera(27.54%) became superior bacteria. The former produces acetic acidand succinic acid during glucose metabolism through EMP pathway(Shah & Collins, 1990), while the latter can produce propionic acid,butyric and valeric acid through EMP pathway at pH 6.0 (Weimer &Moen, 2013). In addition, Acidaminococcus (2.23%) can produce aceticacid, butyric acid and H2 using some ammonia acid (Cook et al., 1994).The relative reactions can be concluded using Eqs. (10)–(12) plus Eqs.(13)–(15).

+ → +C H O 2H 2CH CH COOH 2H O6 12 6 2 3 2 2 (13)

→ + + +3C H O 4CH CH COOH 2CH COOH 2H O 2CO6 12 6 3 2 3 2 2 (14)

+ + → +CH CH COOH 2CO 6H CH CH CH CH COOH 4H O3 2 2 2 3 2 2 2 2

(15)

Fig. 3. Taxonomic classification of main bacterial genera (with relative abun-dance higher than 1% in the inoculum) during anaerobic fermentation of foodwaste under different pH.

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Fig. 4. Metabolic pathways of acidogenic fermentation under different pH.

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3.4. Variation of other parameters and shift of metabolic pathways

In addition to pH, ORP is another key factor related to fermentationstatus (Pei et al., 2011), and extracellular ORP in fermentation liquidcan reflect intracellular ORP since they keep dynamic balance (Graefet al., 1999; Liu et al., 2013). The intracellular ORP determined by thebalance of NAD(P)H/NAD(P)+ (Huang et al., 2017), which has stan-dard potential difference (E0) of −320mV (Liu et al., 2013). Theconversion from glucose to pyruvate generates NADH and decreaseenvironmental ORP. The environmental ORP was the highest at pH 3.2because the decomposition of FW was weakest under this condition.The further conversion from pyruvate to lactic acid, ethanol, propionicacid and butyric acid can re-oxidize NADH to NAD+, but the conversionfrom pyruvate to acetic acid through EMP cannot oxidize NADH andresult in the accumulation of NADH. Hence, high content of acetic acidwas related to low ORP, as shown in Table 3. However, the condition ofpH 4.5 was an exception, and it had higher acetic acid concentrationand also higher ORP than pH 4.7 and 6.0. This is because the formergenerated acetic acid mainly through Bifidus pathway of Bifidobacteria,while the latter two had more acetic acid generation through thepathway of Megasphaera. Bifidus pathway leads to the balance of NADHand NAD+ without the accumulation of NADH. The production fromH+ to H2 can also change ORP (Liu et al., 2013), and thus, high yield ofH2 was related to low ORP.

TAN in fermentation liquid was derived from the decomposition ofprotein. The high TAN at low pH 3.2–4.2 could be attributed to the highORP, which should benefit the degradation of protein (Yin et al., 2016).In addition, LAB can utilize arginine by arginine deiminase and producemore NH3 to optimize intracellular pH to resist acid environment(Castillo Martinez et al., 2013).

The composition of fermentation products was determined by pH,ORP, TAN, etc., but the essential mechanism should be the variation ofmicrobial community and metabolism under different pH conditions.Based on the above analyses and some relative references, the meta-bolic pathways of acidogenic fermentation of FW were concluded cor-responding to different pH (Fig. 4). The cited metabolic pathways thatwas not observed in this study possibly existed under the fermentationenvironment, including ethanol type fermentation through EMPpathway (Ren et al., 1997), ethanol fermentation through Entner-Doudoroff (ED) pathway (Lawford et al., 1988), acetone-butanol-ethanol fermentation through EMP pathway (Wu et al., 2015a), andethanol fermentation through EMP pathway (Lin & Tanaka, 2006). Infact, EMP pathway and pentose phosphate pathway (PPP) always co-exist in microbial metabolic process, and fructose 6-P and pyruvic acidproduced from PPP can be coupled with EMP pathway (Tan et al.,2016).

3.5. Assessment on fermentation degree

For anaerobic fermentation of FW, a key concern is the quantity offermentation product(s) that could be utilized or recovered. Table 4exhibits the ratios of fermentation products in total mass and the fer-mentation degrees, which were expressed as HR and AR. The resultsshowed that pH 3.2–4.5 led to different types of lactic acid fermenta-tion, and the maximum HR and AR occurred at pH 4.5 with high yieldsof lactic acid plus acetic acid and butyric acid. Hence, low pH wassuitable for lactic acid recovery from fermentation liquid due to highpurity, while the fermentation products at pH 4.5 can be used for me-thanogenesis due to the high AR and the complex composition of pro-ducts. The pH 4.7–5.0 led to butyric acid fermentation. The yield ofbutyric acid reached the maximum at pH 4.7, which implied the po-tential of butyric acid recovery. The condition of pH 5.0 resulted in highratios of acetic acid in liquid and hydrogen in gas, and this conditioncan be adopted during simultaneous hydrogen and acid production. Thehighest pH condition 6.0 produce a large quantity of VFAs with a littleof ethanol and lactic acid, which can be also used for biomethane

production. On the whole, AR was adopted to represent the total yieldof target products including lactic acid, ethanol and VFAs, and the threeconditions of pH 4.5, 4.7 and 6.0 performed the best.

HR represents the dissolution of solid organics in FW and impliedthe efficiencies of fermentation and subsequent methanogenesis (if intwo-phase anaerobic digestion). The highest HRCOD occurred at pH 4.5and 4.7 possibly owing to the co-existing of LAB and HPB. The secondbest condition was pH 6.0 and 5.0, and their HRCOD was a little less thanthose at pH 4.5–4.7. Statistical tests indicated that there was no sig-nificant difference between pH 4.5 and 4.7 (p > 0.1) or between pH5.0 and 6.0 (p > 0.1) from the view of point of hydrolysis, while thedifference between the two groups (pH 4.5–4.7 and 5.0–6.0) was sig-nificant (p < 0.01). Jiang et al. (2013) reported that pH 6.0 led to thehighest HR, and (Wu et al., 2015b) found that HR increased sig-nificantly when pH increased from 4.0 to 5.0. However, they did notinvestigate the condition of pH 4.5. HRTOC, that reflected the dissolu-tion of organic carbon, was slightly lower than HRCOD. For the condi-tion of pH 4.7 and 5.0, the difference of about ten percentages indicatedthat there were some carbon-free substances in the liquid after butyricfermentation. The highest HRTOC occurred at pH 4.5 and 6.0, and thefermentation liquid can be used as carbon source for biological waste-water treatment plants. Compared HRTOC and ARTOC, the great differentat pH 5.0 showed the existing of other possible products like acetoneand butanol (Wu et al., 2015a; Zhou et al., 2018). This phenomenonwas consistent with the deduction on other possible metabolic path-ways as mentioned in Fig. 4.

Furthermore, the carbon flow during FW fermentation was calcu-lated (Fig. 5), and the result exhibited the global evolution of organicproducts along with pH variation. On the whole, the transition fromlactic acid to VFAs was very obvious, and the other products includingethanol and CO2 accounted for only a small fraction. For the production

Table 4Ratios of fermentation products and rates of hydrolysis and acidification (thepercentages of lactic acid, ethanol and VFAs indicate their ratios to their sum bymass).

pH 3.2 4.0 4.2 4.5 4.7 5.0 6.0

Lactic acid (%) 86.4 80.5 81 56.7 2.9 6.1 2.4Ethanol (%) 7.9 9.4 8.9 3.3 9.7 12.7 3.3Acetic acid (%) 5.0 3.4 3.4 25 19.3 40.1 16.3Propionic acid (%) 0.4 1.7 1.7 3.8 4.5 0.9 8.9Butyric acid (%) 0.2 3.7 3.3 9.3 56 40.0 39.9Valeric acid (%) 0.1 1.3 1.7 1.9 7.6 0.2 29.2H2 in biogas (%) 8.9 2.9 9.5 32.7 43.7 31.3HRCOD (%) 30.1 42.5 43.2 50.3 50.4 46.5 47.8HRTOC (%) 28.9 39.0 39.2 49.0 40.3 36.9 43.1ARCOD (%) 13.0 34.5 34.6 45.4 45.2 25.5 44.3ARTOC (%) 11.7 30.4 30.6 40.6 35.0 20.6 34.3

Fig. 5. Carbon distribution during anaerobic fermentation of food waste underdifferent pH.

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of lactic acid, pH 4.0–4.2 should be the optimal condition; while pH 6.0resulted in the maximal yield of VFAs. From the aspect of carbon dis-tribution, lactic acid, VFAs, ethanol and CO2 contained most of carbon,but a large quantity (more than 50%) of organic carbon still hide in theundissolved solids of FW. Hence, further improvement of hydrolysisand acidification is much valuable for FW utilization. Nevertheless, theresults provided a clear guideline for FW treatment plants that how toobtain different target products during anaerobic fermentation throughconvenient pH control.

4. Conclusion

The pH condition determined fermentation types and microbialcommunity during anaerobic fermentation of food waste. At pH3.2–4.5, lactic fermentation of Lactobacillus dominated the system, andBifidobacterium also contributed a lot at pH 4.5. The pH 4.7 was theoptimal condition for butyric acid production, and Megasphaera thatconverts lactic acid to butyric acid played a crucial role. The rate ofhydrolysis and acidification reached the maximum at pH 4.5–4.7. Themaximum hydrogen yield was obtained at pH 5.0. The pH 6.0 resultedin mixed acid fermentation with the highest yield of VFAs.

Acknowledgements

Financial support for this project is obtained from the ShenzhenScience and Technology Project (grant numberJCYJ20170307152224920); and the Development and ReformCommission of Shenzhen Municipality (urban water recycling and en-vironment safety program).

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